Myocardial ischemia/reperfusion injury is mediated by mitochondrial dysfunction in which damaged mitochondria utilize oxygen at reperfusion for the generation of reactive oxygen species (ROS) rather than energy. ROS have destructive potential. The most beneficial experimental intervention thus far identified for preventing reperfusion injury is ischemic preconditioning (IPC), which occurs when the myocardium is exposed to brief ischemia-reperfusion events prior to lethal ischemia-reperfusion. Previous work has evaluated single parallel pathways that produce IPC utilizing a linear model of membrane receptor activation leading to cardiac protection; however, no single, unifying mechanism has been proposed for IPC to account for its temporal efficiency (i.e., rapid coupling of plasma membrane to mitochondria) and spatial 3-dimensionality (i.e., simultaneous activation of numerous pathways). An emerging idea in signal transduction (i.e., the existence of multi-protein complexes organized in discrete cellular compartments with dynamic potential) suggests a possible solution to this temporal/spatial conundrum of IPC. Two membrane structural proteins, caveolin-3 (Cav-3) and connexin43 (Cx43), exist in dynamic membrane microenvironments, and provide spatial organization to multi-protein complexes in lipid- rich or gap junctional microdomains of the plasma membrane. Little is known regarding Cav-3 and Cx43 interaction and trafficking in the preconditioned myocardium and the effect of post-translational modifications of these proteins on cardiac protection. Our data suggest a close association of caveolae, caveolin, Cx43, and mitochondria and a possible role for this association in IPC. The aims will address the following specific hypotheses: 1: The post-translational modification of caveolin and Cx43 induced by IPC optimizes caveolae/mitochondria interaction to reduce deleterious ROS generated during ischemic-reperfusion injury. 2: Caveolin and Cx43 are essential for IPC-induced interaction of caveolae and mitochondria. 3: Overexpression of caveolin/Cx43 can augment the interaction of caveolae and mitochondria to mimic IPC. The long-term goal of this work is to identify novel signaling mechanisms relevant to cardiac protection and determine the molecules critical in coordinating a multitude of signaling networks in producing a preconditioning phenotype. PUBLIC HEALTH RELEVANCE Injury form a heart attack is lethal and results in death. Certain proteins found in the heart control the degree of injury. It is the goal of this study to discover key proteins involved in protection against a heart attack injury so that drugs can be appropriately designed.